CN105287547A - Use of Logeracemin A in preparation of anti-hypoxic drug - Google Patents

Abstract

The invention discloses a use of Logeracemin A in preparation of an anti-hypoxic drug. Through the whole and cell-level anoxic model, Logeracemin A anti-hypoxic effects are observed. The observation result shows that Logeracemin A can substantially improve a survival rate and survival time of asphyxia anoxia and acute decompression anoxia mice, reduce myocardial damage of drug specific anoxia mice and produce substantial protection effects on in-vitro culture neonatal rat cardiac myocyte and nerve cell hypoxia damage. The invention provides the use of Logeracemin A in preparation of an anti-hypoxic drug.

Description

Logeracemin A is preparing the application in anti-anoxic medicine

Technical field

The present invention relates to the novelty teabag of compound L ogeraceminA, particularly relate to LogeraceminA and preparing the application in anti-anoxic medicine.

Background technology

Oxygen is the essential condition that the mankind and many biologies are depended on for existence.Hypoxia (Hypoxia) refers to that the oxygen needed for body vital movement can not obtain sufficient supply.Oxygen and hypoxia are the most important key factors of vital movement, are the important topics of life sciences basic theories.The formation of hypoxia can be divided three classes: the first kind is that external environment oxygen content reduces, and makes normal physiological activity process can not absorb enough oxygen, as plateau and aviation anoxia; Equations of The Second Kind refers to because disease etc. causes extraneous normal oxygen amount fully not arrive in body, causes the anoxia of the heart, brain and respiratory system etc.; 3rd class is body activities requisite oxygen consumption, has exceeded the physiology ability of mobilization, causes relative oxygen supply not enough, is common in strenuous exercise and the amount of transfiniting work.Long-term hypoxia is the important hidden danger be detrimental to health, and severe patient can threat to life.Therefore, hypoxia causes the heart, brain and respiratory system equivalent damage to become one of 21 century medical circle subject matter anxious to be resolved.

The compound L ogeraceminA that the present invention relates to is one and delivers (Jin-BiaoXu in 2014, etal., LogeraceminA, anAnti-HIVDaphniphyllumAlkaloidDimerwithaNewCarbonSkelet onfromDaphniphyllumlongeracemosum.J.Am.Chem.Soc.2014, 136, noval chemical compound 7631-7633.), this compound has brand-new framework types, current purposes only relates to anti AIDS virus (Jin-BiaoXu, etal., LogeraceminA, anAnti-HIVDaphniphyllumAlkaloidDimerwithaNewCarbonSkelet onfromDaphniphyllumlongeracemosum.J.Am.Chem.Soc.2014, 136, 7631-7633.), the LogeraceminA that the present invention relates to is belonged to first public preparing the purposes in anti-anoxic medicine, owing to belonging to brand-new structure type, and it is unexpectedly strong for Substituted phenyl-lactic acid, there is not the possibility being provided any enlightenment by other compounds, possesses outstanding substantive distinguishing features, for anti-hypoxia, obviously there is significant progress simultaneously.

Summary of the invention

For above-mentioned prior art, the object of the invention is to find that LogeraceminA has significant oxygen lack resistant function, can be used for preventing and treating anoxia-induced apoptosis disease, thus add the application of LogeraceminA.

LogeraceminA of the present invention is preparing the application in anti-anoxic medicine, is that LogeraceminA is preparing the application prevented and treated in hypoxic damage medicine.Described compound L ogeraceminA structure is as shown in formula I:

The LogeraceminA that the present invention relates to belongs to first public preparing the purposes in anti-anoxic medicine, because framework types belongs to brand-new framework types, and its Substituted phenyl-lactic acid is unexpectedly strong, there is not the possibility being provided any enlightenment by other compounds, possess outstanding substantive distinguishing features, for anti-hypoxia, obviously there is significant progress simultaneously.

Detailed description of the invention

The preparation method of compound L ogeraceminA involved in the present invention is see document (Jin-BiaoXu, etal., LogeraceminA, anAnti-HIVDaphniphyllumAlkaloidDimerwithaNewCarbonSkelet onfromDaphniphyllumlongeracemosum.J.Am.Chem.Soc.2014,136,7631-7633.)

The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not by any restriction of specific embodiment, but be limited by claim.

Embodiment 1: the preparation of compound L ogeraceminA tablet involved in the present invention:

Get 20 g of compound LogeraceminA, add the customary adjuvant 180 grams preparing tablet, mixing, conventional tablet presses makes 1000.

Embodiment 2: the preparation of compound L ogeraceminA capsule involved in the present invention:

Get 20 g of compound LogeraceminA, add prepare capsule customary adjuvant as starch 180 grams, mixing, encapsulatedly makes 1000.

Its pharmaceutically active is further illustrated below by pharmacodynamic experiment.

Experimental example 1: test a mice specificity myocardial ischemia experiment

1, method: 75 kunming mices, body weight (20 ± 2) g.Be divided into 5 groups at random, gastric infusion.First 2 groups give 0.3% sodium carboxymethyl cellulose (CMC-Na) solution, and latter 3 groups give LogeraceminA0.015,0.03gKg respectively ^-1, propranolol hydrochloride 0.03gKg ^-1, after 50min, except the 1st group, equal lumbar injection isoproterenol (ISO) 15mgKg ^-1, after 15min, mice is put into normobaric hypoxia device, record mouse diing time and oxygen consumption.

2, result:

Isoproterenol, by excited heart beta receptor, makes myocardial oxygen consumption increase.This experiment shows, compared with Vehicle controls group, and LogeraceminA0.015,0.03gKg ^-1significantly can resist myocardial oxygen consumption increase (P<0.01) that isoproterenol (ISO) causes, extend the time-to-live (P<0.01) under anoxia in mice air-tight state simultaneously, the results are shown in Table 1.

Table 1LogeraceminA causes the impact (x ± s, n=15) of specificity hypoxia mice on isoproterenol

Compare with matched group, * P<0.01, compare with isoproterenol group, #P<0.01

Test two mice normal pressure asphyxia anoxia experiments

1, method:

60 kunming mices, body weight (20 ± 2) g.Be divided into 4 groups at random, gastric infusion.1st group gives 0.3% sodium carboxymethyl cellulose (CMC-Na) solution, and the 2nd group gives propranolol hydrochloride 0.03gKg ^-1, pro group.3rd, 4 groups give the CMC-Na solution containing LogeraceminA respectively, and concentration is respectively 0.015,0.03gKg _-1.After administration 50min, be placed in wide mouthed bottle and cover tightly bottle stopper (placing 5g sodica calx in bottle).Take respiratory arrest as mark, the record mouse survival time.

2, result:

Compared with Vehicle controls group, LogeraceminA0.015,0.03gKg ^-1make the time-to-live of mice under atmospheric closed condition extend 28.12% and 40.14% respectively, difference has significance (P<0.01, P<0.05).

Test three mice hypobaric hypoxia experiments

1, method:

40 kunming mices, body weight (20 ± 2) g.Be divided into 4 groups at random, gastric infusion.Administration group gives LogeraceminA, and concentration is respectively 0.015,0.03gKg ^-1, matched group gives 0.3%CMC-Na solution, and gavage volume is 2mlKg ^-1.After 50min, administration group and matched group respectively get 5, put into decompressor, stop decompression when 26.7Kpa (be equivalent to height above sea level and be about 10000m), keep this pressure constant, when animal dead 50%, stop decompression immediately, slowly put into air, take out animal, record each group of dead and viable count, repetitive operation is to having tested.

2, result:

LogeraceminA0.015,0.030gKg _-1make the survival rate of mice under hypobaric hypoxia condition be increased to 70%, 80% by 30%, 20% of matched group, difference has significance (P<0.05).

Test the protective effect of four pairs of Myocytes Anoxia damages

1, method:

(1) Neonatal Rat Primary Cardiomyocytes is cultivated: the SD neonatal rat of newborn 1-3d is got ventricular muscles and is cut into about 1mm ₃size piece of tissue, adds 0.25% trypsin-0.02%EDTA, with 37 DEG C at digest.Except digesting supernatant first and discarding, collect each supernatant and stop digestion, collected by centrifugation myocardial cell precipitates, add the DMEM/F-12 culture medium containing 10% hyclone and Brdu (final concentration 0.1mmol/L), blow and beat mixing gently, make cell suspension, be inoculated in culture bottle, in 37 DEG C, 5%CO ₂hatch 70min in incubator, make most non-myocardial infarction adherent.

Draw bottle inner cell suspension counting, adjustment cell concentration is 1 × 10 ⁵individual/ml, is inoculated into 24 orifice plates, every hole 1ml; 96 orifice plates, every hole 200ul.After 48h cell attachment, change the DMEM/F-12 culture medium (not containing Brdu) containing 10% hyclone, within later every 2 days, change liquid 1 time.

(2) Myocytes Anoxia model is set up: get the myocardial cell cultivating 4d, after changing serum-free DMEM/F-12 culture medium continuous culture 12h, (be filled with 95%N in advance with sugar-free D-Hanks liquid ₂-5%CO ₂the saturated 30min of gaseous mixture) substitute normal incubation medium, then rapid immigration by culture plate is connected with 95%N ₂-5%CO ₂in the hypoxia device of gaseous mixture, detect air vent oxygen concentration (<1%) with oxygen analyser, 37 DEG C of anoxias cultivate 6h.

(3) experiment grouping: blank group, anoxia model group, anoxia+LogeraceminAA group (concentration 0.024mg/ml), B group (concentration 0.012mg/ml), C group (concentration 0.006mg/ml) are established in experiment, totally 5 groups, often organize 6 holes.Except blank group, other respectively organize equal anoxic treatment 6h, and Normal group is then in 37 DEG C, 5%CO ₂synchronously 6h is hatched in incubator.

(4) anoxia-induced apoptosis myocardial cell MTT experiment: take out each group of sample, every hole adds 20ulMTT (5g/L), in 37 DEG C, 5%CO ₂continue in incubator to hatch 4h, stop cultivating, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.Add 150ulDMSO jolting 15min, crystallization is fully dissolved, in mensuration wavelength 570nm, reference wavelength 630nm place, measure each hole absorbance (OD) value.

MTT metabolic rate (%)=experimental group OD value/Normal group OD value × 100%

(5) biochemical indicator detects: get each group of cell culture supernatant being incubated at 24 orifice plates, active with colorimetric method for determining LDH, CK, measures SOD in cell active with xanthine oxidase; With MDA content in thiobarbituricacidα-colorimetric method for determining cell, concrete testing process and operational approach are undertaken by test kit description.

2, result:

Cardiac muscle cells because of anoxia sustain damage time, mitochondrial function is abnormal, and oxidation-respiration chain is impaired, and electron transmission blocks, and ATP produces minimizing.Succinate dehydrogenase is one of compound enzyme important in respiratory chain of succinic acid oxidation, the dehydrogenation of catalysis succinic acid can generate Fumaric acid, thus makes FAD accept two hydrogen atoms to generate FADH ₂, and then hydrogen is passed to C _oq, generates C _oqH ₂, continue the electron transfer process of respiratory chain.So, the activity of succinate dehydrogenase, can reflecting myocardium cell hypoxia damage degree.MTT experiment principle, is utilized succinate dehydrogenase can generate the characteristic of the insoluble color products of aubergine by catalysis Thiazolyl blue (MTT) exactly, is reflected the cytoactive of each group by the mensuration of absorbance.This experimental result shows: concentration is respectively 0.024mg/ml, 0.012mg/ml, 0.006mg/mlLogeraceminA, MTT records OD value and is significantly higher than model group, show all significantly to increase cytoactive (P<0.01), illustrate that LogeraceminA to the infringement of anti-hypoxia to myocardial cell succinic dehydrogenase activity, can maintain the respiratory function of Hypoxic cell; When anoxia causes cell membrane damage, in cell, LDH, CK will leak outside, LDH, CK activity in culture medium is caused to raise, this test display 3 dosage groups compared with model group in culture medium LDH, CK activity all obviously reduce, show that LogeraceminA can under Conditions of Acute Hypoxia in Human Body, keep the integrity of myocardial cell membrane, the above results is in table 2.

SOD reflects the ability of cell clearance free radical, MDA reflects Cell membrane lipids Peroxidative damage degree, 3 dosage groups SOD activity rising in cell compared with model group of this test, MDA level produces and declines, in table 3, show the cell membrane oxidative damage that LogeraceminA causes anti-hypoxia by the ability strengthening myocardial cell scavenging free radicals.

Table 2LogeraceminA is on the impact (x ± s, n=6) of the outer leakage quantity of anoxic myocardial LDH, CK

Compare with model group, * * P<0.01, * P<0.05

Table 3LogeraceminA is on the impact (x ± s, n=8) of anoxic myocardial vigor, SOD activity and MDA content

Compare with model group, * * P<0.01, * P<0.05

Conclusion: LogeraceminA can significantly improve survival rate and the time-to-live of asphyxia anoxia and acute decompression hypoxia mice; alleviate the myocardial damage of drug specificity hypoxia mice; to the neonatal rat myocardial cell of In vitro culture and neurocyte anoxia-induced apoptosis, also there is significant protective effect, provide LogeraceminA and preparing the purposes in anti-anoxic medicine.

Claims (1)

1.LogeraceminA the application in anti-anoxic medicine, described compound L ogeraceminA structure is as shown in formula I:

Formula I.